The Cytological Status of Plants Regenerated from Shoot-Meristem Culture of Pisum sativum L

The cytological status of plantlets regenerated from shoot apical meristems of Pisum sativum was investigated. Chromosome counts in root apices of in vitro regenerated plants showed a preponderance of diploid cells. Moreover, the karyotypes of root-tips from plants derived from culture and from normal plants were basically the same. Topics such as the treatment of chromosomal armlength data, simple statistical comparison of samples derived from normal and regenerated plants are discussed.     

Plant Regeneration from in vitro Cultured Anthers of Black Mustard (Brassica nigra Koch)

Anthers of Brassica nigra, excised from fresh as well as cold-pretreated (3 days at 3 ± 2°C) buds cultivated on modified B₅ medium (Gamborg et al. 1968) containing sucrose level varying from 2 % to 10 %, along with 1O^?6M BAP (benzylaminopurine) and 9 × 10^?6M 2,4-D (2,4-dichlorophenoxyacetic acid), developed calli and/or embryos. The latter response was observed only in anthers reared on media containing 6 % or higher levels of sucrose. On media containing two or four per cent sucrose, the anthers produced calli, exclusively. The growth of embryos was inhibited or else they started callusing if left on the media containing higher levels of sucrose. However, on transfer to MS medium (Murashige and Skoog 1962), containing 2 % sucrose, embryos started callusing and subsequently a few secondary embryos differentiated. Such embryos were sub-cultured on MS + 5 × 10^?6M BAP + 2 % sucrose, wherein numerous shoots developed from embryos. The shoots were rooted by transferring to a medium containing 5 × 10^?6M NAA (naphthalene acetic acid). Within two months of culture, some of these plants started flowering in vitro.     

Callus Formation and Plant Regeneration from Protoplasts Derived from Suspension Culture of Rice (Oryza sativa L. cv. 'Taipei 177')

Protoplasts were isolated from young inflorescence-derived suspension cultures of a japonica rice cultivar ‘Taipei 177’. The isolated protoplasts which were cultured either in liquid, agar on Sea plaque agarose underwent sustained division. Maximum plating efficiency of 1.06% occurred in a medium containing macroelements of KM, microelements and vitamins of B5, 0.5 % Sea plaque agarose, 1.0 mg/l of 2,4-D, and glucose as an osmotic stabilizer. Green and albino plants were regenerated from the protocalli in MS semisolid medium containing 4 mg/l BAP, 0.5 mg/l NAA and 500 mg/l casein hydrolysate (MS_18–2).     

Androgenetic response of heterozygous triticale populations using a greenhouse hydroponic system

Production of haploids, followed by chromosome doubling to produce doubled haploids (DH) represents the most rapid means of achieving complete inbreeding. In order to improve the androgenetic responses and maximize the production of green regenerants from selected plants of twenty-five triticale (× Triticosecale, Wittmack) populations (BC₁F₁, TC₁F₁, and F₂) we used a uniform and optimal growth environment for anther donor plants within a greenhouse hydroponic system. Non-orthogonal analysis of deviance showed highly significant differences (p < 0.001) among populations for both induction and regeneration. The overall induction response of the populations was very high with a mean of 50.4 embryoids per 100 anthers plated. Among all tested-populations, M86-6068/TW179//EP80 (TC₁F₁) was the most responsive for both induction and regeneration which could be associated to its Triticum timopheevii cytoplasm. On the other hand, although populations 80465/II83-194 both as BC₁F₁ and F₂ had a high level of induction response, only a few green plants were regenerated. These populations probably possess a partial Secale montanum genome, which could be contributing to the low regeneration ability. In conclusion, the need for optimization of donor plant growth conditions to effectively assess the androgenetic ability of individual populations/lines would be emphasized. This revised version was published online in August 2006 with corrections to the Cover Date.     

Modification of oilseed rape to produce oils for industrial use by means of applied tissue culture methodology

Summary A programme of research was designed to investigate methods for the modification of the fatty acid profiles of high performance lines of oilseed rape (Brassica napus L.) in an attempt to produce lines with enhanced levels of industrially useful fatty acids. The methodology employed to achieve these objectives was based on the exploitation of somaclonal or protoclonal variation, and targeted somatic hybridization using wild cruciferous germplasm as fusion partners.A range of somaclonal lines was produced from shoot regeneration protocols. These lines underwent replicated, randomised glasshouse trials for morphological assessment followed by gas chromatographic analysis to monitor any changes in fatty acid profile. It was found that a small number of lines exhibited potentially useful changes in oleic acid and polyunsaturated fatty acid content. Protoplast regeneration and electrofusion protocols for a range of winter oilseed rape lines were developed, and methods for the isolation and fusion of protoplasts of the wild crucifer Lunaria annua (chosen for its high nervonic acid content) established.Abbreviations GC Gas Chromatography     

Microspore culture is successful in most crop types of Brassica oleracea L.

Summary Microspore culture was shown to be applicable to a broad range of accessions belonging to six horticulturally important crop types of Brassica oleracea: broccoli, white cabbage, cauliflower, savoy cabbage, Brussels sprouts and curly kale. Of 64 accessions tested 86% were responsive. Large genotypic differences were found in number of embryos produced per flower bud, and in frequency and mode of regeneration of plants from embryos. B. oleracea was characterized by a strong asynchrony of microspore development within single buds. Microspore populations optimal for culture contained a large proportion (10–40%) of binucleate pollen. An initial high temperature treatment was essential for microspore embryogenesis. Growth conditions of the donor plants during inflorescence formation were less critical.     

Efficient In Vitro Shoot-regeneration Systems in Cassava (Manihot esculenta Crantz)

Two different protocols for in vitro regeneration of cassava using zygotic embryos and nodal axillary meristems have been developed. In both cases, buds were regenerated directly from excised explants without an intervening callus phase after a two-step culture procedure. In cotyledonary explants derived from zygotic embryos, prolific shoot formation occurred within 2—3 weeks on MS medium supplemented with 0.5—5 mg/1 BAP alone or in combination with 0.1 mg/1 NAA. Nodal explants with axillary meristems derived from aseptically grown seedlings or stem cuttings were used to initiate a round compact bulb-like structure on MS medium containing 10 mg/1 BAP. These latter structures, when cultured on MS medium supplemented with 0.1 mg/1 NAA, 1 mg/1 BAP and 0.1 mg/1 GA₃, produced multiple shoots. Somatic embryos isolated at the globular/torpedo stage from zygotic embryo explants were also capable of multiple shoot production on medium with 1 mg/1 BAP. Rooting of regenerated shoots exceeded 95 % in phytohormone-free MS medium. No change in their ploidy levels was observed. Therefore, the protocols developed should be of use in the particle gun and Agrobacterium-mediated genetic transformation of cassava.     

影响籼粳稻杂交F1花药培养效果的因素分析

以津稻291/IRBB60籼粳杂交F1、反交F1及其亲本的花药为外植体,对诱导和分化培养基、激素配比及有机附加物和高温预培养时间等影响花药培养效果的因素进行研究。结果表明,在SK3基本培养基中添加2 mg/L 2,4-D,1 mg/L NAA和1 mg/L KT比只添加2 mg/L 2,4-D的愈伤诱导率高0.67～2.78个百分点;在诱导培养基中添加水解酪蛋白能明显提高正交F1的愈伤诱导率;MS 1 mg/L NAA 2 mg/L 6-BA 0.5 mg/L KT分化培养基得到了较高的绿苗分化率;正交F1的愈伤诱导率、绿苗分化率和花培力显著高于反交F1;30℃高温预培养36 h,正交F1花培力达到最高值。     

Spontaneous induction of tetraploidy in hop using adventitious shoot regeneration method

In the process of breeding triploid hop cultivars, diploid varieties need to be raised to the tetraploid level. Existing methods are predominantly based on treatment of the apices with antimitotic substances, in vivo or in vitro , resulting in a reported maximum of 26% tetraploids and a large proportion (over 60%) of mixoploids. This study was set-up to test whether an adventitious shoot regeneration protocol can be used as an alternative. In the first part of the experiments, regeneration media and a choice of explants (internodes, petioles and leaf discs) were optimized using three hop cultivars with putatively low, medium or high adventitious shoot regeneration capacity. On an optimized induction medium supplemented with 6 mg/l N ⁶-(2-isopentenyl) adenine (2iP), a regeneration rate up to 17.7% was obtained from cultured internodes, even of the most recalcitrant 'Aurora', while the regeneration rate of the most responsive 'Tettnanger' was 56.9%. Flow cytometric analysis of ploidy level revealed a high frequency of tetraploid induction, the highest (58.6%) being in 'Savinjski golding'. In 616 regenerants of all three cultivars, 30.4% were found to be tetraploid, the others were diploid. Cytokinin content was found to be of minor importance in tetraploid induction. The high frequency of spontaneous tetraploid induction and, in particular, the complete absence of mixoploids makes this protocol an alternative to the currently established methods of chromosome doubling based on the use of antimitotic substances. The occurrence of a high proportion of tetraploid adventitious regenerants in hop is discussed in view of certain other biotechnological applications.     

大麦成熟胚胚性愈伤组织的高频诱导和植株再生

大麦成熟胚的初代愈伤组织是水质、柔软的非胚性愈伤组织，在继代培养中能以一定频率出现胚性愈伤组织。本文研究了基因型、有机添加物和胚切割等因素对大麦胚性愈伤组织诱导频率的影响。结果表明：供试的１０个大麦品种在胚性愈伤组织诱导率和植株再生率上有显著差异；培养基中添加水解酷蛋白、Ｌ－脯氨酸以及提高维生素Ｂ１和肌醇的浓度可以促进胚性愈伤组织的形成；胚芽段比全胚或胚根段更易形成胚性愈伤组织。胚性愈伤组织转入无